Name: protein purification-free method of binding affinity determination by microscale thermophoresis
Uploaded: 2013-08-15T13:00:00
Description: Watch this Scientific Journal Video about Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis at JoVE.com

这部分工作是由院内研究项目由美国国立卫生研究院，美国国家癌症研究所癌症研究中心; NCI和黑腹滨鹬治疗之间的合作研究协议，美国癌症协会授予IRG 97-152-17 OT和联邦基金支持美国国家癌症研究所，美国国立卫生研究院，根据合同HHSN26120080001E。

1。细胞裂解液的制备该协议的目的是，粘附细胞，任何GFP融合蛋白表达。所需的细胞数可以从低至10 6到多达20×10 6细胞，这取决于蛋白质表达的水平上。例如，裂解的HEK细胞过表达GFP-STAT3的准备处理10 T75烧瓶中生长的细胞裂解液用1毫升至70％附近汇合。然而，这种裂解液稀释150倍，以提供最佳的荧光水平MST实验。根据调查的性质和细胞内的蛋白质的本地化强?...

<ol>
	<li>Lea, W. A., Simeonov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+polarization+assays+in+small+molecule+screening.">Fluorescence polarization assays in small molecule screening.</a> Expert. Opin. Drug Discov. 6, 17-32 (2011).</li><li>Willander, M., Al-Hilli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+biomolecules+using+surface+plasmons.">Analysis of biomolecules using surface plasmons.</a> Methods Mol. Biol. 544, 201-229 (2009).</li><li>Hulme, E. C., Trevethick, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand+binding+assays+at+equilibrium:+validation+and+interpretation.">Ligand binding assays at equilibrium: validation and interpretation.</a> Br. J. Pharmacol. 161, 1219-1237 (2010).</li><li>Ghai, R., Falconer, R. J., Collins, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+isothermal+titration+calorimetry+in+pure+and+applied+research--survey+of+the+literature+from+2010.">Applications of isothermal titration calorimetry in pure and applied research--survey of the literature from 2010.</a> J. Mol. Recognit. 25, 32-52 (2012).</li><li>Vuignier, K., Schappler, J., Veuthey, J. L., Carrupt, P. A., Martel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-protein+binding:+a+critical+review+of+analytical+tools.">Drug-protein binding: a critical review of analytical tools.</a> Analytical and Bioanalytical Chemistry. , 398-3953 (2010).</li><li>Lebowitz, J., Lewis, M. S., Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+analytical+ultracentrifugation+in+protein+science:+a+tutorial+review.">Modern analytical ultracentrifugation in protein science: a tutorial review.</a> Protein Sci. 11, 2067-2079 (2002).</li><li>Duhr, S., Braun, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+molecules+move+along+a+temperature+gradient.">Why molecules move along a temperature gradient.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 19678-19682 (2006).</li><li>Zillner, K., Jerabek-Willemsen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+thermophoresis+as+a+sensitive+method+to+quantify+protein:+nucleic+acid+interactions+in+solution.">Microscale thermophoresis as a sensitive method to quantify protein: nucleic acid interactions in solution.</a> Methods Mol. Biol. 815, 241-252 (2012).</li><li>Wienken, C. J., Baaske, P., Rothbauer, U., Braun, D., Duhr, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-binding+assays+in+biological+liquids+using+microscale+thermophoresis.">Protein-binding assays in biological liquids using microscale thermophoresis.</a> Nat. Commun. 1, 100 (2010).</li><li>Stark, G. R., Darnell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+JAK-STAT+pathway+at+twenty.">The JAK-STAT pathway at twenty.</a> Immunity. 36, 503-514 (2012).</li><li>Ehret, G. B., Reichenbach, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+binding+specificity+of+different+STAT+proteins.+Comparison+of+in+vitro+specificity+with+natural+target+sites.">DNA binding specificity of different STAT proteins. Comparison of in vitro specificity with natural target sites.</a> J. Biol. Chem. 276, 6675-6688 (2001).</li><li>Yang, J., Stark, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+unphosphorylated+STATs+in+signaling.">Roles of unphosphorylated STATs in signaling.</a> Cell Res. 18, 443-451 (2008).</li><li>Timofeeva, O. A., Chasovskikh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+unphosphorylated+STAT3+transcription+factor+binding+to+DNA.">Mechanisms of unphosphorylated STAT3 transcription factor binding to DNA.</a> J. Biol. Chem. 287, 14192-14200 (2012).</li><li>Timofeeva, O. A., Tarasova, N. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT3+suppresses+transcription+of+proapoptotic+genes+in+cancer+cells+with+the+involvement+of+its+N-terminal+domain.">STAT3 suppresses transcription of proapoptotic genes in cancer cells with the involvement of its N-terminal domain.</a> Proc. Natl. Acad. Sci. U.S.A. , (2013).</li><li>Patterson, G., Day, R. N., Piston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein+spectra.">Fluorescent protein spectra.</a> J. Cell Sci. 114, 837-838 (2001).</li></ol>

蛋白表达和纯化是一个费 ​​力和昂贵的步骤，然而，有必要由目前使用最多的方法测定相互作用的K D。 MST的应用可避免蛋白质纯化，从而大大简化和加快相互作用的定量表征。难以表达和纯化的蛋白质，如膜蛋白和转录因子的情况下特别显着的优点。 的主要限制和要求的MST是作为与绿色荧光蛋白融合的蛋白质的表达能力。然而，构建表达GFP融合人类和小鼠蛋白质?...

亲和力的分子间相互作用的定量表征是非常重要的生物医学研究在许多领域。约束力的解离常数（K D）是必不可少的，不仅在药物发现的，但也是一个重要的参数，表征任何生物系统中的任何二进制的相互作用。生化用于检测蛋白质-蛋白质相互作用的方法，如免疫沉淀和酵母双杂交筛选，不告知我们这些交互是多么紧张，而亲和定义，这是否存在特别复杂的给定条件下体内 。在药物发现过程中，结合法的发展是必要的，往往是最耗时的步骤之一。 杀敌测定最常用的方法包括荧光偏振，表面等离子体共振（SPR）技术，2放射配体结合，3温滴定量热平衡态，4透析（ED），超滤（UF），第5和超速离心（UC）。他们需要纯化的靶蛋白的数量显著。微量热泳（MST）是一个迅速发展的方法，该方法检测在一个微小的温度梯度定向的分子运动。任何更改的运动沿温度梯度的相对变化的生物分子导致的水合壳8，7 MST是用来确定结合亲和力，并已申请了荧光标记的蛋白质或荧光配体的配体结合研究靶蛋白。 9 MST允许直接在溶液中的相互作用的测量，而不需要固定到表面上（无固定技术）。实际上，任何有约束力的MST信号的变化是伴随着的大小变化，虽然从系统到系统的显着不同。对于检测MST分子运动，他们必须荧光NT。这种方法的主要限制，可以变成优势。如果在任何系统中作为GFP融合蛋白的表达，它是唯一的荧光分子，从而可以从细胞溶胞产物不经分离或无细胞表达系统研究。代约束条件允许以最少的文物的细胞裂解液中的主要挑战。在这里，我们描述了一个协议，可用于许多可溶性蛋白和膜蛋白的细胞裂解物的制备和MST实验。 STAT蛋白是潜在的胞质转录因子激活的酪氨酸磷酸化胞外信号响应和参与许多生物过程，包括免疫，造血，炎症，和发展10，在哺乳动物，STAT家族由STAT 1，2，3，4的，5A，5B，和6。所有激活的统计数据绑定到相同的DNA序列，所谓的气体图案，IFN-γ的激活序列。然而，transcri不同统计的ptional效果有很大的不同。11尽管在许多病理过程中的参与和广泛的研究，产生超过17,000出版物，K D STAT相互作用不同的DNA序列尚未确定。只有相对亲和力不同的统计变种天然气动机特点。11蛋白表达和纯化困难是表征统计的DNA结合选择性的主要障碍。虽然大多数研究都集中在“激活”的统计资料，这成了转录因子酪氨酸磷酸化，非磷酸化的统计（U-统计）转录调控作用的作用正在迅速崛起。 然而 ，这些机制的了解甚少，不清楚是否实际上U-统计与DNA结合，或通过与其他转录因子的相互作用行事。最近，我们已经表明，U-STAT3可以绑定到DNA顺序CES不同气图案具有更高的亲和力。13的发现具有重大意义，为我们理解这一重要蛋白质的生物学功能。我们应用微尺度热泳，确定相对亲和力STAT3气和AT丰富的寡核苷酸Ş+100（ 图4）。几乎相同的协议被用于不同的的STAT3的配位体，脂肽类抑制剂的结合在K D测定14否将关联的转录因子，作为阴性对照，使用GFP-STAT1结合从而证实选择性相互作用时，可检测到的。 14

<table border="1">
	<tbody>
 <tr>
 <td>Name of the Reagent/material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>RIPA buffer</td>
 <td>Millipore</td>
 <td>20-188</td>
 <td>Other manufacturer's buffers work as well</td>
 </tr>
 <tr>
 <td>Protease inhibitors cocktail</td>
 <td>Sigma-Aldrich</td>
 <td>P2714</td>
 <td></td>
 </tr>
 <tr>
 <td>Monolith NT.115</td>
 <td>NanoTemper Technologies GmbH</td>
 <td>G008</td>
 <td></td>
 </tr>
 <tr>
 <td>Monolith NT.115 Capillary Tray</td>
 <td>NanoTemper Technologies GmbH</td>
 <td>T001</td>
 <td></td>
 </tr>
 <tr>
 <td>Monolith NT.115 Standard Treated Capillaries</td>
 <td>NanoTemper Technologies GmbH</td>
 <td>K002</td>
 <td></td>
 </tr>
 <tr>
 <td>NT Control software</td>
 <td>NanoTemper Technologies GmbH</td>
 <td>2.0.2.29</td>
 <td></td>
 </tr>
 <tr>
 <td>NT Analysis software</td>
 <td>NanoTemper Technologies GmbH</td>
 <td>1.4.27</td>
 <td></td>
 </tr>
 <tr>
 <td>Table-top refrigerated centrifuge</td>
 <td>Eppendorf </td>
 <td>5417R</td>
 <td>Other microtube refrigerated centrifuges</td>
 </tr>
 <tr>
 <td>Protein LoBind Tube 0.5 ml</td>
 <td>Eppendorf </td>
 <td>22431064</td>
 <td></td>
 </tr>
	 </tbody>
 </table>

protein purification-free method of binding affinity determination by microscale thermophoresis

蛋白质相互作用的定量表征是在几乎任何领域的生命科学，特别是药物发现至关重要。 K D测定所需要的现有的方法获得纯化的蛋白质，还代可以是费时和昂贵的。我们已经开发出一种测定结合亲和力的微尺度热泳（MST）协议，允许未经纯化的目标蛋白从细胞裂解液。该方法涉及过表达GFP的融合蛋白，在非变性条件下的细胞裂解。该方法应用到STAT3-GFP瞬时表达在HEK293细胞中的第一次，以确定具有不同序列的寡核苷酸的充分研究的转录因子的亲合性的。该协议是简单的，并且可以有各种不同的应用研究小分子，肽，DNA，RNA的蛋白质的相互作用，和对roteins。

测量非磷酸化STAT3的寡核苷酸结合蛋白的亲合性。 STAT3-GFP表达HEK293细胞，使用荧光标记的STAT3的DNA结合测定作为源。用RIPA缓冲液（20×10 6细胞/ ml）制备细胞裂解液。对于结合的研究，裂解物稀释150倍MST DNA的结合缓冲液中的荧光蛋白的结合反应（约20纳米），以提供最佳水平。未转染的HEK293细胞中已被用于评估的背景荧光，这被证明是不可探测的，即使在非...

Watch this Scientific Journal Video about Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis at JoVE.com

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

可广泛用于微型热泳（MST）无需纯化从细胞裂解液中的目标蛋白的结合亲和力的测定。该协议涉及过表达GFP融合蛋白，在非变性条件下的细胞裂解，MST信号检测在不同浓度的配体的存在下。

蛋白质纯化方法测定结合亲和力微尺度热泳

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

Research

JoVE Journal

Biology

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

微量法测定蛋白浓度使用NanoDrop 2000C分光光度计

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

科研

生物学

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

正确的蛋白浓度的动力学结构与功能分析和亲和力测定的重要性

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

脂质囊泡介导的亲和层析法，使用磁性细胞分选（LIMACS）：一种新的方法来分析蛋白质 - 脂质相互作用

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Biochemical Reconstitution of Steroid ReceptorÃƒÂ¢Ã¢â€šÂ¬Ã‚Â¢Hsp90 Protein  Complexes and Reactivation of Ligand Binding

类固醇受体的生化重建•Hsp90的蛋白质复合物和配体激活绑定

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

由一个小型的双特异性亲和标签的推动正交蛋白质纯化

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

视黄醇运输的血浆视黄醇结合蛋白的膜受体的实时分析

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

一个协议，用于噬菌体展示和亲和选择以重组蛋白诱饵

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...